PROJECT SUMMARY/ABSTRACT Decades of research have been unable to fully explain why women have a higher incidence of rheumatoid ar- thritis (RA) compared to men or why the post-partum period is associated with a markedly increased incidence of RA. Formation of anti-citrullinated (cit) protein antibodies (ACPA) is a well-established early step in the devel- opment of RA, and recent reports support that mucosal sites are involved in ACPA formation during a pre-clinical phase of RA development. Studies to date have focused on the lung, gingival or gut mucosae in RA, but these studies have not helped to explain sex differences in RA. As such, the proposed project will investigate the novel hypothesis that the female genital tract and lactating mammary tissue are mucosal sites of ACPA generation unique to women. In the R61 phase, cervicovaginal fluid (CVF) will be collected from pre-menopausal women with RA, first-degree relatives (FDRs) of RA patients and healthy controls. Breast milk will be collected from post- partum women with RA, FDRs and controls. CVF, breast milk and blood will be tested for IgG-ACPA and IgA- ACPA, including both cit and arginine-containing proteins/peptides that can establish cit-specificity. Total IgG and IgA will be measured to calculate ACPA/Ig ratios at each site. CVF and breast milk samples with high ACPA levels will undergo immunoprecipitation, and cit-peptides bound in immune complexes will be identified by mass spectrometry. It is expected that a portion of pre-menopausal women will have elevated CVF and breast milk cit- specific ACPA, higher ACPA/Ig ratios in CVF and breast milk compared to blood and immune complexes in CVF and breast milk that bind cit-peptides, thereby confirming ACPA generation at these mucosal sites. In addition, plasmablasts from breast milk will be quantified by flow cytometry, and a subset of sorted plasmablasts will undergo antibody repertoire sequencing. Presence of multiple clonal families in breast milk plasmablasts will support an active immune response in lactating mammary tissue associated with ACPA. If ACPA generation at these sites is confirmed in the R61 phase, then the R33 phase will confirm that ACPA generation is caused by inflammation originating at these mucosal sites. Specifically, human CVF, breast milk and blood collected in the R61 phase will be tested for inflammatory cytokines and correlated with ACPA. It is expected that RA-related cytokines will be associated with ACPA at these sex-specific mucosal sites. In addition, HLA-DR4 transgenic and wild type mice will undergo intravaginal immunization with cit-peptides and native peptide controls. A subset will also become pregnant. CVF, breast milk and blood in these mice will be tested for ACPA. It is expected that cit-peptides will directly induce inflammation and ACPA generation in the CV mucosa. In breast milk, it is ex- pected that ACPA will be induced by naturally occurring cit-peptides. Importantly, understanding the generation of ACPA at these mucosal sites specific to women could revolutionize the way we think about the etiology of sex differences in RA and how we approach personalized treatment and prevention of RA.